G protein-coupled receptors (GPCRs) are cell surface proteins that translate hormone or ligand binding into intracellular signals. GPCRs have been found in all animals, insects, and plants studied to date. GPCR signaling plays a pivotal role in regulating various physiological functions including phototransduction, olfaction, neurotransmission, vascular tone, cardiac output, digestion, pain, and fluid and electrolyte balance. Although they are involved in various physiological functions, GPCRs share a number of common structural features. They contain seven membrane domains bridged by alternating intracellular and extracellular loops and an intracellular carboxyl-terminal tail of variable length.
The magnitude of the physiological responses controlled by GPCRs is linked to the balance between GPCR signaling and signal termination. The signaling of GPCRs is controlled by a family of intracellular proteins called arrestins. Arrestins bind activated GPCRs, including those that have been agonist-activated and especially those that have been phosphorylated by G protein-coupled receptor kinases (GRKs).
Receptors, including GPCRs, have historically been targets for drug discovery and therapeutic agents because they bind ligands, hormones, and drugs with high specificity. Approximately fifty percent of the therapeutic drugs in use today target or interact directly with GPCRs. See e.g., Jurgen Drews, (2000) “Drug Discovery: A Historical Perspective,” Science 287:1960–1964.
Although only several hundred human GPCRs are known, it is estimated that upwards of a thousand GPCRs exist in the human genome. Of these known GPCRs, many are orphan receptors that have yet to be associated with a function or a ligand.
There is a need for accurate, easy to interpret methods of detecting G protein-coupled receptor activity and methods of assaying GPCR activity. One method, as disclosed in Barak et al., U.S. Pat. Nos. 5,891,646 and 6,110,693, uses a cell expressing a GPCR and a conjugate of an arrestin and a detectable molecule, the contents of which are incorporated by reference in their entirety.
The majority of the existing methods for identifying GPCR antagonists are dependent on the presence of agonist. Assays for identifying compounds that prevent the activation of GPCRs typically require that the GPCR is first activated in order to identify interfering compounds. For receptors with known agonists, these agonists are currently used to activate these receptors. However, many GPCRs are orphan receptors with no known ligand or agonist.
One method, as disclosed in Pausch et al., WO 00/12704, uses GPCRs with constitutively activating mutations that permit detection of the receptors' functional activity in the absence of activating ligands. In Pausch et al. modifications are made to the GPCR that result in a constitutively active receptor. The constitutively active GPCR mutants of Pausch et al. have elevated intrinsic activity compared to wild type receptors and interact with and activate intracellular heterotrimeric G proteins in an agonist-independent manner. The method of Pausch et al., although agonist-independent, utilizes constitutively active GPCR mutants.
The agonist-dependence of GPCR assays continues to be a problem because antagonist discovery for orphan receptors is typically dependent on the prior discovery of agonist or ligand. Therefore, there is a need to provide additional methods of GPCR activation that are not dependent on agonist.